Vaccines against escherichia coli O157 infection

ABSTRACT

This invention relates to conjugates of the O-specific polysaccharide of  E. coli  O157 with a carrier, and compositions thereof, and to methods of using of using of these conjugates and/or compositions thereof for eliciting an immunogenic response in mammals, including responses which provide protection against, or reduce the severity of, bacterial infections. More particularly it relates to the use of polysaccharides containing the tetrasaccharide repeat unit: (→3)-α-DGalpNAc-(1→2)-α-D-PerpNAc-(1→3)-α-L-Fucp-(1→4)-β-D-Glcp-(1→), and conjugates thereof, to induce serum antibodies having bactericidal (killing) activity against hemolytic-uremic syndrome (HUS) causing  E. coli , in particular  E. coli  O157. The conjugates, and compositions thereof, are useful as vaccines to induce serum antibodies which have bactericidal or bacteriostatic activity against  E. coli , in particular  E. coli  O157, and are useful to prevent and/or treat illnesses caused by  E. coli  O157.  
     The invention further relates to the antibodies which immunoreact with the O-specific polysaccharide of  E. coli  O157 and/or the carrier, that are induced by these conjugates and/or compositions thereof. The invention also relates to methods and kits using one or more of the polysaccharides, conjugates or antibodies described above.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of prior U.S. application Ser. No.09/744,289, filed Aug. 1, 2001, which is the national stage under § 371of PCT Application No. PCT/US98/14976, filed Jul. 20, 1998, published inEnglish under PCT Article 21(2). The prior applications are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to conjugates of the O-specific polysaccharide ofShiga toxin-producing bacteria, such as E. coli O157, with carrier, andcompositions thereof, and to methods of using of these conjugates and/orcompositions thereof for eliciting an immunogenic response in mammals,including responses which provide protection against, or reduce theseverity of, bacterial infections. More particularly it relates to theuse of polysaccharides containing the tetrasaccharide repeat unit:(→3)-α-D-GalpNAc-(1→2)-α-D-PerpNAc-(1→3)-α-L-Fucp-(1→4)-β-D-Glcp-(1→),and conjugates thereof, to induce serum antibodies having bactericidal(killing) activity against E. coli, in particular E. coli O157. Theconjugates, and compositions thereof, are useful as vaccines to induceserum antibodies which have bactericidal or bacteriostatic activityagainst E. coli, in particular E. coli O157, and are useful to preventand/or treat illnesses caused by E. coli O157.

The invention further relates to the antibodies which immunoreact withthe O-specific polysaccharide of E. coli O157 and/or the carrier, thatare induced by these conjugates and/or compositions thereof. Theinvention also relates to methods and kits for detection,identification, and/or diagnosis of E. coli O157, using one or more ofthe polysaccharides, conjugates or antibodies described above.

BACKGROUND

The most successful of all carbohydrate pharmaceuticals so far have beenthe carbohydrate-based, antibacterial vaccines [1]. The basis of usingcarbohydrates as vaccine components is that the capsular polysaccharidesand the O-specific polysaccharides on the surface of pathogenic bacteriaare both protective antigens and essential virulence factors. The firstsaccharide-based vaccines contained capsular polysaccharides ofPneumococci: in the United States a 14-valent vaccine was licensed in1978 followed by a 23-valent vaccine in 1983. Other capsularpolysaccharides licensed for human use include a tetravalentmeningococcal vaccine and the Vi polysaccharide of Salmonella typhi fortyphoid fever. The inability of most polysaccharides to elicitprotective levels of anti-carbohydrate antibodies in infants and adultswith weakened immune systems could be overcome by their covalentattachment to proteins that conferred T-cell dependent properties [2].This principle led to the construction of vaccines against Haemophilusinfluenzae b (Hib) [3] and in countries where these vaccines areroutinely used, meningitis and other diseases caused by Hib have beenvirtually eliminated [4]. Extension of the conjugate technology to theO-specific polysaccharides of Gram-negative bacteria has provided a newgeneration of glycoconjugate vaccines that are undergoing various phasesof clinical trials [5].

Escherichia coli O157:H7, an emerging infectious agent, was firstrecognized as a human pathogen in 1983 [6]. Diseases caused by thispathogen have subsequently been recognized worldwide [7]. Infection withE. coli O157 causes a spectrum of illnesses with high morbidity andmortality, ranging from watery diarrhea to hemorrhagic colitis and theextraintestinal complication of hemolytic-uremic syndrome (HUS). HUS canlead to acute renal failure requiring dialysis, and in children andinfants this complication has a considerable mortality. In some studies,E. coli O157 was the most common cause of dysentery in patients seen inhospital clinics [8].

E. coli strains associated with HUS produce at least one toxin identicalto the exotoxin of Shigella dysenteriae serotype 1, referred to hereinas Shiga toxin 1 (Stx1). This toxin has been variously referred to inthe literature as Vero cytotoxin 1 (VT1), Shiga-like toxin 1 (SLT-I),and Shiga toxin 1(Stx-I or Stx1). In some cases a second toxin(variously referred to as VT2, SLT-II, Stx-II, or Stx2), structurallyand functionally related to Stx 1 and having a cross-reactive A subunit,is also produced. Infection with Stx-producing organisms has beencorrelated with HUS, and E. coli O157:H7 is a common serotype thatproduces these toxins. However, strains of E. coli O157 without Stx havebeen isolated from patients with hemorrhagic colitis.

The pathogenicity of E. coli O157 has been compared to that of Shigelladysenteriae type 1 [9, 10]. Both E. coli O157 and S. dysenteriae type Isecrete almost identical exotoxins (Stx1 or Stx2) and cause bloodydiarrhea, with its complications, only in humans. Antibiotic treatmentdoes not ameliorate the course of enteritis caused by E. coli O157, andit may in fact increase the incidence of HUS caused by E. coli and S.dysenteriae type 1 [11,12]. Unlike S. dysenteriae type 1, which isconfined to humans, E. coli O157:H7 lives in cattle and in otherdomesticated animals without causing symptoms. The feces of infectedanimals serve as a source of E. coli O157 infection in humans, throughcontamination of drinking water and meat.

Most adults have low or nondetectable levels of serum antibodies to E.coli O157 C-SP and to Shiga toxins. High levels of O-SP antibodies andlow or nondetectable levels of antitoxin are regularly found followinginfection with E. coli O157 and the subsequent complication HUS. It isnot known whether immunity follows infection with this pathogen.

Although there is no consensus on the host factors that might conferimmunity to E. coli O157, the O-specific polysaccharide portion of thelipopolysaccharides of the similar genus Shigella have emerged aspossible protective antigens [13,14]. These polysaccharides were shownto be essential for the virulence of Shigella, and it is nowwell-established that the protection is serotype specific. Since eachserotype is characterized by a distinct O-specific polysaccharide, it isfair to say that protection against E. coli O157 is also O-specificpolysaccharide specific. The safety and immunogenicity of a proteinconjugate of the O-specific polysaccharides of S. sonnei, S. flexneri2a, and S. dysenteriae type 1 has been demonstrated in human volunteers,and preliminary clinical trials have established the efficacy of thesevaccines [9, 15, 16, 17].

The immunogenicity of saccharides, alone or as protein conjugates, isrelated to several variables: 1) species and the age of the recipient;2) molecular weight of the saccharide; 3) density of the saccharide onthe protein; 4) configuration of the conjugate (single vs. multiplepoint attachment); and 5) the immunologic properties of the protein.

Because high molecular weight polysaccharides can induce the synthesisof antibodies from B-cells alone, they are described as T-independentantigens. Three properties of polysaccharides are associated withT-independence; 1) their repetitive polymeric nature, which results inone molecule having multiple identical epitopes; 2) a minimum molecularweight that is related to their ability to adhere to and cross-linkmembrane-bound IgM receptors, resulting in signal transduction andantibody synthesis; and 3) resistance to degradation by mammalianenzymes. Most capsular polysaccharides are of comparatively highmolecular weight (≧150 kD), and elicit antibodies in older children andin adults but not in infants and young children. O-SPs are of lowermolecular weight (≦100 kD), and may be considered to be haptens becausethey combine with antibody (are antigenic) but do not elicit antibodysynthesis (are not immunogenic). The immunogenicity of O-SPs asconjugates may be explained by two factors: 1) the increase in molecularweight that allows the O-SP to adhere to a greater number ofmembrane-bound IgM and induce signal transduction to the B-cell; and 2)their protein component, which is catabolized by the O-SP stimulated Bcell resulting in a peptide-histocompatibility II antigen signal to Tcells.

Synthesis of conjugates for use as vaccines in humans has specialconsiderations. LPS is not suitable for parenteral administration tohumans because of toxicity mediated by the lipid A domain. Usually, O-SPis prepared by treatment of LPS with either acid or hydrazine in orderto remove fatty acids from lipid A. The resultant products retain thecore region and the O-SP with its heterogeneous range of molecularweights (Mr). Conjugates are prepared by schemes that bind the carrierto the O-SP at multiple sites along the O-SP, or attempt to activate oneresidue of the core region.

In the case of E. coli O157, vaccine development has been hinderedbecause there is little information about mechanisms of immunity [9],and there are no valid animal models for diseases caused by E. coliO157[10].

There have been some efforts to date to attempt to obtain effectivevaccine compositions against E. coli. See, e.g., Cryz et al. (U.S. Pat.No. 5,370,872), which describes the isolation of O-SP derived from LPSof 12 serotypes of E. coli and their covalent linkage to P. aeruginosatoxin A as a carrier protein [18]. The twelve monovalent conjugates werecombined to form a polyvalent vaccine, which was described as being safeand immunogenic in both rabbits and humans when administered byinjection. An antibody response to both the O-SP and toxin A moietieswas reported, and protection of rabbits against E. coli sepsis wasdemonstrated upon passive immunization with the resulting IgGantibodies. However, neither bactericidal activity of the antibodies norprotection after vaccination with the conjugates was shown, andantibodies against E. coli strain O157 and protection against E. coliO157 infection are not mentioned.

Because anti-LPS or anti-O-SP antibody-mediated protection is likely tobe serotype-specific, it is unlikely that the polyvalent vaccinedescribed in U.S. Pat. No. 5,370,872 would induce a significant level ofantibodies against E. coli O157 O-SP or LPS. There remains a need,therefore, for compositions and methods of inducing a significant levelof antibodies against E. coli O157. There also remains a needcompositions and methods for inducing antibodies which have bactericidalactivity against E. coli O157, and which also prevent or ameliorate HUS.

BRIEF DESCRIPTION OF THE INVENTION

It is an object of the invention to produce antigens based on theO-specific polysaccharide of Shiga toxin-producing bacteria,particularly E. coli O157, conjugated with a carrier, and compositionsthereof, and to methods of using of these conjugates and/or compositionsthereof for eliciting an immunogenic response in mammals, includingresponses which provide protection against, or reduce the severity of,bacterial infections. More particularly, it is an object of theinvention to provide conjugates having polysaccharides containing thetetrasaccharide repeat unit:(→3)-α-D-GalpNAc-(1→2)-α-D-PerpNAc-(1→3)-α-L-Fucp-(1→4)-β-D-Glcp-(1→),and compositions thereof, to induce serum antibodies having bactericidal(killing) activity against E. coli, in particular E. coli O157. Theconjugates, and compositions thereof, are useful as vaccines to induceserum antibodies which have bactericidal or bacteriostatic activityagainst against E. coli, in particular E. coli O157, and are useful toprevent and/or treat illnesses caused by E. coli O157.

It is yet another object of the present invention to provide conjugatesof E. coli O157 O-SP bound to the non-toxic B-subunit of Shiga toxin 1(StxB1), or mutated non-toxic holotoxin of Shiga toxin 1 or Shiga toxin2. These conjugates have the advantage of inducing both (1) serum IgGanti-O157-LPS with bactericidal activity, and (2) neutralizingantibodies to Shiga toxin 1 or Shiga toxin 2 (Stx1 or Stx2)[19,20,21].

It is also an object of the invention to provide antibodies whichimmunoreact with the O-specific polysaccharide of E. coli O157 and/orthe carrier, that are induced by these conjugates and/or compositionsthereof. Such antibodies may be isolated, or may be provided in the formof serum containing these antibodies.

It is also an object of the invention to provide a method for thetreatment or prevention of E. coli O157 infection in a mammal, byadministration of compositions containing the antibodies of theinvention, or serum containing the antibodies of the invention.

The invention also provides methods and kits for identifying, detecting,and/or diagnosing E. coli O157 infection or colonization using theantibodies which immunoreact with the O-specific polysaccharide of E.coli. The invention also relates to methods and kits for identifying,detecting and/or diagnosing the presence of Shiga toxins 1 or 2.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides conjugates of an E. coli O157 O-specificpolysaccharide covalently bound, either directly or through a linker, toa carrier, and compositions thereof. The present invention alsoencompasses mixtures of such conjugates and compositions thereof. In apreferred embodiment, the carrier is the non-toxic B subunit of Shigatoxin 1 or 2 (StxB1, StxB2), or a non-toxic mutant of Stx1 or Stx2holotoxin. In yet another preferred embodiment, the particular E. coliO157-Stx conjugate is part of a composition containing O-SP-carrierconjugates from other E. coli strains that commonly cause HUS, to form amultivalent vaccine for broad coverage against HUS. Hyperimmune plasmacontaining both anti-LPS and neutralizing antibodies to Stxs areexpected to provide protective and therapeutic effects in at-riskindividuals and in patients during outbreaks.

The invention also provides methods of using these conjugates orcompositions thereof to induce in mammals, in particular, humans, theproduction of antibodies which immunoreact with the O-specificpolysaccharide of E. coli O157. In the preferred embodiment, antibodieswhich immunoreact with Shiga toxin 1 or Shiga toxin 2 are also produced.The antibodies which immunoreact with the O-specific polysaccharide ofE. coli O157 are useful for the identification, detection, and/ordiagnosis of E. coli O157 colonization and/or infection. Antibodieswhich have bactericidal or bacteriostatic activity against E. coli/O157are useful to prevent and/or treat illnesses caused by E. coli O157.Antibodies which immunoreact with Shiga toxins 1 and 2 are useful toneutralize Shiga toxins 1 and 2, and either decrease the incidenceand/or severity of hemolytic-uremic syndrome, or prevent the increase ofits incidence and/or severity, in established infections.

Pharmaceutical compositions of this invention are capable, uponinjection into a human of an amount containing 25 μg of E. coli O157O-specific polysaccharide, of inducing in the serum bactericidalactivity against E. coli O157, such that the serum kills, in thepresence of complement, 50% or more of E. coli O157 at a serum dilutionof 1300:1 or more. Preferred compositions can induce serum bactericidalactivity against E. coli O157 such that the serum kills 50% or more ofE. coli O157 at a serum dilution of 32,000:1 or more, and the mostpreferred compositions can induce serum bactericidal activity against E.coli O157 such that the serum kills 50% or more of E. coli O157 at aserum dilution of 64,000:1 or more. The O-SP conjugate vaccines of thisinvention are designed to induce serum IgG antibodies that willinactivate an inoculum of E. coli O157 at the entrance of the jejunumbefore an infection is established.

The invention also provides a saccharide-based vaccine, which isintended for active immunization for prevention of E. coli O157infection, and for preparation of immune antibodies as a therapy,preferably for established infections. The vaccines of this inventionare designed to confer specific preventative immunity against infectionwith E. coli O157, and to induce antibodies specific to E. coli O157O-SP and LPS. The E. coli O157 vaccine is composed of non-toxicbacterial components, suitable for infants, children of all ages, andadults.

The conjugates of this invention, and/or compositions thereof, as wellas the antibodies thereto, will be useful in increasing resistance to,preventing, ameliorating, and/or treating E. coli O157 infection inhumans, and in reducing or preventing E. coli O157 colonization inhumans.

This invention also provides compositions, including but not limited to,mammalian serum, plasma, and immunoglobulin fractions, which containantibodies which are immunoreactive with E. coli O157 O-SP, and whichpreferably also contain antibodies which are immunoreactive with Shigatoxins 1 or 2, in particular with the B subunit of Shiga toxins 1 or 2.These compositions, in the presence of complement, have bacteriostaticor bactericidal activity against E. coli O157. These antibodies andantibody compositions are useful to prevent, treat, or ameliorateinfection and disease caused by the microorganism. The invention alsoprovides such antibodies in isolated form.

High titer anti-O157 sera, or antibodies isolated therefrom, could beused for therapeutic treatment for patients with E. coli O157 infectionor hemolytic-uremic syndrome (HUS). Antibodies elicited by the O-SPconjugates of this invention may be used for the treatment ofestablished E. coli O157 infections, and are also useful in providingpassive protection to an individual exposed to E. coli O157.

The present invention also provides diagnostic tests and/or kits for E.coli O157 infection and/or colonization, using the conjugates and/orantibodies of the present invention, or compositions thereof.

The present invention also provides an improved method for synthesizingan O-SP peptide conjugate, particularly the E. coli O157 O-SP conjugatedto the B subunit of Shiga toxin 1 or 2 (Stx1 or Stx2), or to a mutant,non-toxic Stx1 or Stx2 holotoxin.

A number of primary uses for the conjugates of this invention areenvisioned. The E. coli LPS-protein conjugates of this invention, andthe antibodies they induce, are expected to be useful for severalpurposes, including but not limited to:

-   -   1) a vaccine for high-risk groups (children under 5 and senior        citizens);    -   2) high-titered globulin for plasmapheresis, for prophylaxis and        treatment of E. coli O157-infected patients; and    -   3) diagnostic reagents for detecting and/or identifying E. coli        O157.

The invention is intended to be included in the routine immunizationschedule of infants and children, and in individuals at risk for E. coliO157 infection. It is also planned to be used for intervention inepidemics caused by E. coli O157. Additionally, it is may be used as acomponent of a multivalent vaccine for E. coli O157 and other entericpathogens, useful for example for the routine immunization of infants.The invention is also intended to prepare antibodies with bacteriostaticbactericidal activity toward E. coli O157, for therapy of establishedinfection. The invention is also intended to provide a diagnostic testfor E. coli O157 infection and/or colonization.

Definitions

Galp=galactosaminopyranosyl; Perp=perosaminopyranosyl;Fucp=fucopyranosyl; Glcp=glucopyranosyl.

As used herein, the term “O-SP” when used alone refers generically toO-specific polysaccharide, whether produced by acidolysis orhydrazinolysis of lipopolysaccharide. When used in designatingconjugates, however (e.g. O-SP-rEPA, DeA-LPS-rEPA, etc.) these productsare differentiated by use of the term “O-SP” for O-specificpolysaccharide produced by acidolysis, and the term “DeA-LPS” forO-specific polysaccharide produced by hydrazinolysis.

As used herein, the terms “immunoreact” and “immunoreactivity” refer tospecific binding between an antigen or antigenic determinant-containingmolecule and a molecule having an antibody combining site, such as awhole antibody molecule or a portion thereof.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules.Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and portions of animmunoglobulin molecule, including those portions known in the art asFab, Fab′, F(ab′)₂ and F(v), as well as chimeric antibody molecules.

Polymeric Carriers

Carriers are chosen to increase the immunogenicity of the polysaccharideand/or to raise antibodies against the carrier which are medicallybeneficial. Carriers that fulfill these criteria are described in theart [22, 23, 24, 25]. A polymeric carrier can be a natural or asynthetic material containing one or more functional groups, for exampleprimary and/or secondary amino groups, azido groups; or carboxyl groups.The carrier can be water soluble or insoluble.

Water soluble peptide carriers are preferred, and include but are notlimited to natural or synthetic polypeptides or proteins, such as bovineserum albumin, and bacterial or viral proteins or non-toxic mutants orpolypeptide fragments thereof, e.g., tetanus toxin or toxoid, diphtheriatoxin or toxoid, Pseudomonas aeruginosa exotoxin or toxoid, recombinantPseudomonas aeruginosa exoprotein A, pertussis toxin or toxoid,Clostridium perfringens and Clostridium welchii exotoxins or toxoids,mutant non-toxic Shiga toxin holotoxin, Shiga toxins 1 and 2, the Bsubunit of Shiga toxins 1 and 2, and hepatitis B surface antigen andcore antigen.

Examples of water insoluble carriers include, but are not limited to,aminoalkyl SEPHAROSE, e.g., aminopropyl or aminohexyl SEPHAROSE(Pharmacia Inc., Piscataway, N.J.), aminopropyl glass, and the like.Other carriers may be used when an amino or carboxyl group is added, forexample through covalent linkage with a linker molecule.

Methods for Attaching Polymeric Carriers

Methods for binding a polysaccharide to a protein are well known in theart. For example, a polysaccharide containing at least one carboxylgroup, through carbodiimide condensation, may be thiolated withcystamine, or aminated with adipic dihydrazide, diaminoesters,ethylenediamine and the like. Groups which can be introduced by suchknown methods include thiols, hydrazides, amines and carboxylic acids.Thiolated and aminated intermediates are stable, and may be freeze driedand stored cold. Thiolated intermediates may be covalently linked to apolymeric carrier containing a sulfhydryl group, such as a2-pyridyldithio group. Aminated intermediates may be covalently linkedto a polymeric carrier containing a carboxyl group through carbodiimidecondensation. See for example reference [26], where 3 different methodsfor conjugating Shigella O-SP to tetanus toxoid are exemplified. Becausethe methods of the present invention better preserve the nativestructure of the antigen, they are preferred over methods which oxidizethe polysaccharide with periodate [18].

The polysaccharide can be covalently bound to a carrier with or withouta linking molecule. To conjugate without a linker, for example, acarboxyl-group-containing polysaccharide and an amino-group-containingcarrier are mixed in the presence of a carboxyl activating agent, suchas a carbodiimide, in a choice of solvent appropriate for both thepolysaccharide and the carrier, as is known in the art [25]. Thepolysaccharide is often conjugated to a carrier using a linkingmolecule. A linker or crosslinking agent, as used in the presentinvention, is preferably a small linear molecule having a molecularweight of about 500 or less, and is non-pyrogenic and non-toxic in thefinal product form, for example as disclosed in references [22-25].

To conjugate with a linker or crosslinking agent, either or both of thepolysaccharide and the carrier may be covalently bound to a linkerfirst. The linkers or crosslinking agents are homobifunctional orheterobifunctional molecules, e.g., adipic dihydrazide, ethylenediamine,cystamine, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP),N-succinimidyl-N-(2-iodoacetyl)-β-alaninate-propionate (SIAP),succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC),3,3′-dithiodipropionic acid, and the like. Also among the class ofheterobifunctional linkers area omega-hydroxy and omega-amino alkanoicacids.

More specifically, attachment of the E. coli O157 O-specificpolysaccharide to a protein carrier can be accomplished by methods knownto the art. In a preferred embodiment, the attachment is accomplished byfirst cyanating the O-specific polysaccharide with a cyanylationreagent, such as cyanogen bromide, N-cyano-N,N,N-triethylammoniumtetrafluoroborate, 1-cyano-4-(N,N-dimethylamino)pyridinetetrafluoroborate, or the like. Several such cyanylation reagents areknown to those skilled in the art [27]. The resulting cyanated E. coliO157 O-specific polysaccharide may then be reacted with a linker, suchas a dicarboxylic acid dihydrazide, preferably adipic acid dihydrazide,so as to form a hydrazide-functionalized polysaccharide. Thishydrazide-functionalized polysaccharide is then coupled to the carrierprotein by treatment with a peptide coupling agent, preferably awater-soluble carbodiimide such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide methiodide, or the like.

More preferably, the cyanated E. coli O157 O-specific polysaccharide isdirectly reacted with the carrier protein, without introduction of alinker. It has been found, surprisingly, that, in the exemplifiedconjugates, elimination of the customary linker provides a moreeffective immunogen in the case of the E. coli O157 O-specificpolysaccharide.

Regardless of the precise method used to prepare the conjugate, afterthe coupling reactions have been carried out the unbound materials areremoved by routine physicochemical methods, such as for example gelfiltration or ion exchange column chromatography, depending on thematerials to be separated. The final conjugate consists of thepolysaccharide and the carrier bound directly or through a linker.

Dosage for Vaccination

The present inoculum contains an effective, immunogenic amount of apolysaccharide-carrier conjugate of this invention. The effective amountof polysaccharide-carrier conjugate per unit dose sufficient to inducean immune response to E. coli O157 depends, among other things, on thespecies of mammal inoculated, the body weight of the mammal, and thechosen inoculation regimen, as is well known in the art. Inoculatypically contain polysaccharide-carrier conjugates with concentrationsof polysaccharide from about 1 micrograms to about 10 milligrams perinoculation (dose), preferably about 3 micrograms to about 100micrograms per dose, and most preferably about 5 micrograms to 50micrograms per dose.

The term “unit dose” as it pertains to the inocula refers to physicallydiscrete units suitable as unitary dosages for mammals, each unitcontaining a predetermined quantity of active material (polysaccharide)calculated to produce the desired immunogenic effect in association withthe required diluent.

Inocula are typically prepared as solutions in physiologically tolerable(acceptable) diluents such as water, saline, phosphate-buffered saline,or the like, to form an aqueous pharmaceutical composition. Adjuvants,such as aluminum hydroxide, may also be included in the compositions.

The route of inoculation may be intramuscular, subcutaneous or the like,which results in eliciting antibodies protective against E. coli O157.In order to increase the antibody level, a second or booster dose may beadministered approximately 4 to 6 weeks after the initial injection.Subsequent doses may be administered as indicated herein, or as desiredby the practitioner.

Antibodies

An antibody of the present invention in one embodiment is characterizedas comprising antibody molecules that immunoreact with E. coli O157 O-SPor LPS.

An antibody of the present invention is typically produced by immunizinga mammal with an immunogen or vaccine containing an E. coli O157polysaccharide-protein carrier conjugate to induce, in the mammal,antibody molecules having immunospecificity for the immunizingpolysaccharide. Antibody molecules having immunospecificity for theprotein carrier, such as the B subunit of Shiga toxins 1 or 2, will alsobe produced. The antibody molecules may be collected from the mammaland, optionally, isolated and purified by methods known in the art.

Human or humanized monoclonal antibodies are preferred, including thosemade by phage display technology, by hybridomas, or by mice with humanimmune systems. The antibody molecules of the present invention may bepolyclonal or monoclonal. Monoclonal antibodies may be produced bymethods known in the art. Portions of immunoglobulin molecules, such asFabs, may also be produced by methods known in the art.

The antibody of the present invention may be contained in blood plasma,serum, hybridoma supernatants and the like. Antibody-containing serum ofthis invention will be capable of killing, in the presence ofcomplement, 50% of E. coli O157 at a serum dilution of 1300:1 or more,preferably will do so at a dilution of 32,000:1 or more, and mostpreferably will be capable of killing 50% of E. coli O157 at a dilutionof 64,000:1 or more.

Alternatively, the antibodies of the present invention are isolated tothe extent desired by well known techniques such as, for example, ionchromatography or affinity chromatography. The antibodies may bepurified so as to obtain specific classes or subclasses of antibody suchas IgM, IgG, IgA, IgG₁, IgG₂, IgG₃, IgG₄ and the like. Antibodies of theIgG class are preferred for purposes of passive protection. Theantibodies of the present invention have a number of diagnostic andtherapeutic uses. The antibodies can be used as an in vitro diagnosticagents to test for the presence of E. coli O157 in biological samples orin meat and meat products, in standard immunoassay protocols. Suchassays include, but are not limited to, agglutination assays,radioimmunoassays, enzyme-linked immunosorbent assays, fluorescenceassays, Western blots and the like. In one such assay, for example, thebiological sample is contacted with first antibodies of the presentinvention, and a labeled second antibody is used to detect the presenceof E. coli O157 to which the first antibodies have bound.

Such assays may be, for example, of direct format (where the labeledfirst antibody is reactive with the antigen), an indirect format (wherea labeled second antibody is reactive with the first antibody), acompetitive format (such as the addition of a labeled antigen), or asandwich format (where both labeled and unlabelled antibody areutilized), as well as other formats described in the art.

The antibodies of the present invention are also useful in preventionand treatment of infections and diseases caused by E. coli O157.

In providing the antibodies of the present invention to a recipientmammal, preferably a human, the dosage of administered antibodies willvary depending upon such factors as the mammal's age, weight, height,sex, general medical condition, previous medical history and the like.

In general, it is desirable to provide the recipient with a dosage ofantibodies which is in the range of from about 1 mg/kg to about 10 mg/kgbody weight of the mammal, although a lower or higher dose may beadministered. The antibodies of the present invention are intended to beprovided to the recipient subject in an amount sufficient to prevent, orlessen or attenuate the severity, extent or duration of the infection byE. coli O157. Antibodies which immunoreact with Shiga toxin 1 or 2 areintended to be provided to the recipient subject in an amount sufficientto prevent, or lessen or attenuate the severity, extent or duration ofthe infection by Shigatoxin producing organisms, such as E. coli strainsO157, O111, O26, and O17.

The administration of the agents of the invention may be for either“prophylactic” or “therapeutic” purpose. When provided prophylactically,the agents are provided in advance of any symptom. The prophylacticadministration of the agent serves to prevent or ameliorate anysubsequent infection. When provided therapeutically, the agent isprovided at (or shortly after) the onset of a symptom of infection. Theagent of the present invention may, thus, be provided prior to theanticipated exposure to E. coli O157 (or other Shiga toxin producingbacteria), so as to attenuate the anticipated severity, duration orextent of an infection and disease symptoms, after exposure or suspectedexposure to these bacteria, or after the actual initiation of aninfection.

For all therapeutic, prophylactic and diagnostic uses, thepolysaccharide-carrier conjugates of this invention, as well asantibodies and other necessary reagents and appropriate devices andaccessories may be provided in kit form so as to be readily availableand easily used.

The following examples are exemplary of the present processes andincorporate suitable process parameters for use herein. These parametersmay be varied, however, and the following should not be deemed limiting.

EXAMPLES Example 1 Conjugation of E. coli O157 O-SP with VariousPolypeptides

O157 LPS were detoxified by hydrolysis with acetic acid (designatedO-SP) or with hydrazine (designated DeA-LPS) and then covalently boundto Clostridium welchii exotoxin C (Pig Bel toxoid [CW]), Pseudomonasaeruginosa recombinant exoprotein A (rEPA), or bovine serum albumin(BSA) [8]. These E. coli O157:H7 polysaccharide-protein conjugates weregiven the following designations:

-   -   O-SP-BSA₁    -   O-SP-BSA₂    -   DeA-LPS-BSA    -   O-SP-CW    -   DeA-LPS-CW    -   O-SP-rEPA    -   DeA-LPS-rEPA₁    -   DeA-LPS-rEPA₂

Mice were immunized with these conjugate compositions containing 2.5 ugof polysaccharide, with booster injections, and the determination ofantibody levels and bactericidal antibody titers in mice weredetermined. Geometric mean antibody level (ELISA units) andimmunoglobulin class composition of LPS antibodies elicited by E. coliO157-rEPA conjugates in mice are shown in Table 1. TABLE 1Immunoglobulin class composition of LPS antibodies elicited by E. coliO157-rEPA conjugates in mice Geometic mean antibody level (ELISA units)(25^(th)-75^(th) centiles) Immunogen After 1^(st) injection After 2^(nd)injection After 3^(rd) injection IgG O-SP-rEPA 0.08 (0.05-0.10) 2.50*(1.06-4.79)  6.26** (3.37-9.6) DeA-LPS-rEPA₁ 0.07 (0.04-0.13) 1.37*(0.50-2.63) 4.49*** (1.49-16.4) DeA-LPS-rEPA₂ 0.07 (0.06-0.07) 0.66*(0.07-3.73)  5.10** (2.23-10.0) IgM O-SP-rEPA 0.53 (0.36-0.72)  0.51(0.31-1.12)    0.38 (0.22-0.59) DeA-LPS-rEPA₁ 0.11 (0.04-0.34)  0.32(0.08-0.89)    0.94 (0.28-2.94) DeA-LPS-rEPA₂ 0.09 (0.06-0.11)  0.32(0.06-1.53)    0.28 (0.21-0.45)a. IgG and IgM components of the hyperimmune O157 sera (see Materialsand Methods) were used as standards and assigned a value of 100 ELISA Ueach. Injection of O-SP, DeA-LPS, or saline did not elicit detectableantibodies.*P < 0.01 when compared with the value for O-SP-rEPA after the firstinjection;**P > 0.02 when compared with the value for the same immunogen after thesecond injection;***P < 0.07 when compared with the value for the same immunogen afterthe second injection.

Bactericidal activity of serum LPS antibodies elicited in mice byimmunization with heat-killed E. coli O157:H7 or O-specificpolysaccharide-protein conjugates are shown in Table 2 below: TABLE 2Bactericidal activity of serum LPS antibodies elicited in mice byimmunization with heat-killed E. coli O157:H7 or O-specificpolysaccharide-protein conjugates Antibody titer Reciprocal (ELISAunits) bacterial Vaccine^(a) Total IgG IgM titer^(b) Expt 1 O-SP-CW79.25 100 DeA-LPS-CW 15.1 >100 DeA-LPS-CW 19.4 80 E. coli O157:H7 100.035 Expt 2 DeA-LPS-rEPA 18.8 0.07 320 DeA-LPS-rEPA 56.8 0.33 640DeA-LPS-rEPA 32.8 0.45 640 O-SP-rEPA 18.6 0.44 640 O-SP-rEPA 15.8 0.59640^(a) E. coli O157:H7 is pooled hyperimmune sera from mice injected withheat-killed E. coli O157. All other sera were from individual mice takenafter the third conjugate injection. Serum dilutions were mixed with anequal volume of ˜10³ E. coli O157:H7 organisms per ml and complement.^(b)The reciprocal bactericidal titer is expressed as the highest serumdilution yielding 50% killing. Absorption with LPS or DeA-LPS removedall of the bactericidal activity from sera from conjugate-injected miceand 90% from the hyperimmune sera prepared by injection of heat-killedE. coli O157.

Example 2 Conjugation of E. coli O157 I-SP with rEPA; Preparation ofVaccine Compositions

As discussed above, O-SP of E. coli O157, prepared by acetic acidhydrolysis, and DeA-LPS O157, prepared by hydrazinolysis, have beenpreviously described. Conjugates of these polysaccharides to rEPA (O-SPO157-rEPA, DeA-LPS O157-₁, and DeA-LPS O157-rEPA₂) were prepared by thepublished procedure [8]. These conjugates were approved forinvestigation by the NIH (OH94-CH-N040), the FDA (BB-IND-5528) and theInstitutional Review Board, Carolinas Medical Center, Charlotte, N.C.(08-94-08B). Pyrogen, sterility and safety testing of the finalcontainers were performed by the Center for Biologics Evaluation andResearch, FDA. All three conjugates elicited serum IgG anti-LPS withbactericidal activity when injected by a clinically relevant scheme anddosage in mice [8].

Clinical Protocol

Volunteers of either gender and any ethnic group between ages 18 and 44years were recruited from the staff of Carolinas Health Care System andthe city of Charlotte, N.C. Exclusion criteria were: pregnancy orplanned pregnancy in the next six months, positive stool culture for E.coli O157 or a history of hemorrhagic colitis, chronic disease includingHIV 1, hepatitis or inflammatory bowel disease, acute illness includingdiarrhea, taking controlled substances, hospitalization within the year,taking regular medications, participation in another research protocolduring the next two months, abnormal liver function test or havingreceived cholera vaccine [32, 28]. After giving Informed Consent, amedical history and physical examination were performed and blood wasobtained for assay of HIV 1, hepatitis b surface antigen, pregnancytest, liver function tests (LFT), antibodies to E. coli O157 LPS and P.aeruginosa exotoxin A (ETA) and a culture of the stool for E. coli O157.Eighty-seven volunteers were determined healthy and randomized into 3groups of 29 to receive a injection of 0.5 ml of one of the experimentalvaccines containing 25 μg of O-SP. Injections were deliveredintramuscularly into the deltoid muscle. The volunteers were observedfor 30 minutes after vaccination. Temperature and local or systemicreactions were recorded at 6, 24, 48 and 72 hours following vaccination.

All volunteers returned at 1, 4 and 26 weeks following vaccination for ahealth history and reaction, and blood was drawn. LFTs were performed,total protein/albumin), total bilirubin/direct and indirect, alkalinephosphatase (AP), SGOT (AST), SGPT (ALT), and GGT at each visit.Volunteers who had abnormal LFT levels at one week had repeated LFTtests at subsequent visits. Serum was collected for LPS and ETA antibodyassays. Stool cultures for E. coli O157 were obtained prior to and 4 and26 weeks following vaccination. E. coli O157 LPS and P. aeruginosaexotoxin A (ETA) antibodies of the volunteers were determined by ELISA[8].

Statistical Methods

Antibody levels are expressed as geometric means (GM). Levels below thesensitivity of ELISA were assigned the value of one-half of that level.Comparison of GM was performed with either the two-sided t-test, pairedt-test or the Wilcoxon test where appropriate.

Results—Clinical Responses

One volunteer reported 3-6 cm diameter of erythema at the injection sitewithin 24 hours following vaccination; one reported 1-3 cm and onereported >6 cm. Four volunteers reported erythema and induration after72 hours observation: one (1-3 cm), two (3-6 cm) and one (>6 cm); allerythemas resolved by the 17th day.

Six volunteers (6.9%) had asymptomatic elevations (up to 35% above thenormal range) of one or more serum LFT following vaccination. Four ofthese 6 volunteers had mild elevation of LDH and/or AP that returned tonormal at 4-5 weeks. One volunteer had a serum bilirubin of 2.2 mg/dl(normal 1.5 mg/dl) with indirect bilirubin of 1.9 mg/dl at four weeks,and normal values at 14 weeks. Another volunteer had ALT (SGPT) and GGTevaluations of 33% and 26% respectively at four weeks, and elevations13% and 47% respectively at 24 weeks following vaccination.

Ninety percent of volunteers reported oral temperatures less than 37.2°C. at different observation times post-vaccination. The remainder of thevolunteers reported oral temperatures 37.2-38° C. with symptoms of acuterespiratory infections.

There was no significant correlation between the reportedpost-vaccination observations and the lots of vaccine administered andno volunteer was hospitalized during the study.

One recipient of DeA-LPS O157-rEPA₁ had a stool culture positive for E.coli O157 at the 1 week post-vaccination visit. This volunteer had noadverse reactions following vaccination and no complaints throughout thestudy, and subsequent stool cultures were negative for E. coli O157.

Results—Antibody Levels (Tables 3a and 3b)

IgG: Pre-vaccination GM IgG anti-LPS levels in the three groups were lowand similar. One week after vaccination, 71/87 (82%) responded with a≧4-fold rise. Four weeks after vaccination, there were further rises inGM levels in all three groups (p<0.0001): all vaccinees responded with a≧4-fold rise over the 1 week level. The GM levels in recipients ofO-SP-rEPA were slightly higher than in those injected with either of thetwo DeA-LPS-rEPA conjugates (61.9 vs. 46.3 NS, 61.9 vs. 36.3, p<0.05).At 26 weeks, the GM levels of the 3 groups were similar (32.8, 31.2,33.1, NS). Although the decline from the four week level was significantfor each group (p<0.05), the levels at 26 weeks were higher than thoseat one week following vaccination in all three groups (32.8, 31.2, 33.1vs. 7.93, 5.73, 4.12, p<0.01); and 97% of volunteers had ≧10-fold higherlevels at 26 weeks than their pre-injection levels. Within the 25-75percentile range, geometric mean titers were increased 68-fold to132-fold after 4 weeks, and the overall result for the three conjugatesat 4 weeks was a 93-fold increase in geometric mean titer. At 26 weeks,the results were increases of 61-fold to 70-fold, and 64-fold increaseoverall for all conjugates. The volunteer who had a stool culturepositive for E. coli O157 at 1 week had IgG anti-LPS levels atpre-immunization, 1-, 4-, and 26-week post-immunization of 0.81, 1.15,7.73 and 7.01 respectively, that are lower than the GM of all 3 groups.

IgM: Each conjugate elicited a significant rise in IgM anti-LPS at the 4and 26 weeks intervals (p<0.0001). O-SP-rEPA elicited the highest levelat each post vaccination interval but the difference was significantonly at 4 weeks (32.8 vs. 18.1, 19.1, p<0.05). At the 4 week interval,there was a >4-fold rise in 61/87 (70%) and in 34/86 (39.5%) at 26 weekscompared to pre-vaccination levels. There was a significant decrease inserum IgM anti-LPS at 26 weeks in all of the three groups (p<0.02) butthere were no significant differences in the GM levels among the threegroups. The volunteer who had a stool culture positive for E. coli O157at 1 week had a pre-immunization anti-LPS IgM level which was relativelyhigh (11.9). The IgM levels declined 1,4 and 26 weeks post-immunization(7.04, 10.6 and 5.94 units, respectively). These levels are lower thanthe GM of the three groups.

IgA: Pre-vaccination levels of IgA anti-LPS were low. Similar to IgG andIgM anti-LPS, about 90% of the volunteers responded with ≧4-fold rise inIgA anti-LPS at one week, and 99% at four weeks (p<0.001). IgA anti-LPSGM levels declined to about 70% of the levels at the 4 week interval.TABLE 3A Geometric mean titers of serum IgG, IgM, and IgAlipopolysaccharide (LPS) antibodies elicited in volunteers by injectionof E. coli O157 O-SP-rEPA conjugates. ELISA units (25^(th)-75^(th)percentiles) Conjugate Preimmune 1 Week 4 Weeks 26 Weeks IgG O-SP-rEPA0.47 (0.3-0.7) 7.93 (2.8-24) 61.9 (40-91) 32.8 (23-50) DeA-LPS-rEPA₁0.51 (0.3-0.9) 5.73 (1.8-22) 46.3 (22-84) 31.2 (12-61) DeA-LPS-rEPA₂0.54 (0.3-0.9) 4.12 (2.2-6.0) 36.6 (20-76) 33.1 (15-57) IgM O-SP-rEPA8.10 (4.0-14) 32.8 (23.51) 64.7 (47-109) 28.6 (17-44) DeA-LPS-rEPA₁ 7.19(3.1-12) 19.1 (9.2-29) 43.5 (13-56) 22.5 (11-34) DeA-LPS-rEPA₂ 7.41(4.6-13) 18.1 (10-27) 42.7 (26-73) 25.3 (17-35) IgA O-SP-rEPA 0.06(0.0-0.1) 0.98 (0.5-2.4) 1.73 (1.0-2.5) 1.17 (0.9-2.1) DeA-LPS-rEPA₁0.06 (0.0-0.1) 0.58 (0.3-0.8) 1.26 (0.6-3.7) 1.01 (0.5-1.9)DeA-LPS-rEPA₂ 0.07 (0.0-0.1) 0.90 (0.4-1.8) 2.13 (1.2-4.9) 1.40(1.0-2.5)NOTE:High-titered postvaccination sera were used as standards. IgG, IgM, andIgA were assigned value of 100 ELISA units. Each group had 29volunteers.

TABLE 3B Fold increases in geometric mean titers of serum IgG, IgM, andIgA lipopolysaccharide (LPS) antibodies elicited in volunteers. -foldincrease in 25^(th)-75^(th) percentiles Ab class Conjugate 1 Week 4Weeks 26 Weeks IgG O-SP-rEPA 17 132 70 DeA-LPS-rEPA₁ 11 91 61DeA-LPS-rEPA₂ 7.6 68 61 Geometric mean 11 93 64 IgM O-SP-rEPA 4.0 8.03.5 DeA-LPS-rEPA₁ 2.7 6.0 3.1 DeA-LPS-rEPA₂ 2.4 5.8 3.4 Geometric Mean3.0 6.5 3.3 IgA O-SP-rEPA 16 29 20 DeA-LPS-rEPA₁ 9.7 21 17 DeA-LPS-rEPA₂13 30 20 Geometric Mean 13 26 19NOTE:High-titered postvaccination sera were used as standards. IgG, IgM, andIgA were assigned value of 100 ELISA units. Each group had 29volunteers.Results—Serum Bactericidal Activity (Table 4)

Serum from high-responding volunteers (above the 75th percentile) wasdiluted serially and the diluted samples were analyzed for their abilityto kill E. coli O157:H7. Pre-vaccination sera had no detectablebactericidal activity against E. coli O157:H7. The three conjugateselicited serum bactericidal activity that roughly correlated with theserum IgG and IgM anti-LPS antibody levels.

The results in Table 4 are those for serum from high-respondingvolunteers. Typically, the bactericidal titer (reciprocal dilution) for50% killing ranged from about 2400 to about 32000. TABLE 4 Bactericidalactivity (reciprocal titer) of serum anti-lipopolysaccharide (LPS)antibodies elicited in volunteers by injection of E. coli O157 O-SP-rEPAconjugates. Antibody level (ELISA units) Bactericidal Conjugate IgG IgMtiter* Preimmune 0.21 2.92 0 Preimmune 0.84 9.1 0 O-SP-rEPA 120.1354.2 >6.4 × 10⁴ O-SP-rEPA 251.9 112.9   1.3 × 10⁴ O-SP-rEPA 156.3183.6 >1.3 × 10³ DeA-LPS-rEPA₁ 231.4 59.9 >6.4 × 10⁴ DeA-LPS-rEPA₂ 77.568.2   1.3 × 10⁴*Expressed as reciprocal of highest serum dilution yielding 50% killing.Results—Serum Anti-P. aeruginosa Exotoxin A (Table 5)

Most volunteers had low or non-detectable ETA antibodies in theirpre-vaccination sera. All three conjugates elicited significantincreases in GM IgG anti-ETA at the 1-week (p<0.002) and 4-week(p<0.001) intervals. At 26 weeks, the GM levels declined to thoseobserved one week after vaccination. There were no statisticallysignificant differences in the GM IgG anti-ETA at each bleeding intervalamong the three groups. TABLE 5 Serum antibodies to Pseudomonasaeruginosa exotoxin A (ETA) elicited by Escherichia coli 0157 O-specificpolysaccharide-rEPA conjugates in volunteers GM antibody level (ELISAUnits*) Conjugate n Preimmune 1 week 4 weeks 26 weeks O-SP-rEPA 29 0.290.93 1.90 0.88 DeA-LPS-rEPA₁ 29 0.39 0.91 1.48 0.87 DeA-LPS-rEPA₂ 290.29 0.65 0.93 0.67*A high titered volunteer serum was used as a standard and assigned avalue of 100 ELISA Units.

Example 3 Conjugation of E. coli O157 O-SP with STXB1 and Preparation ofVaccine Compositions

E. coli O157 O-SP was prepared by treatment of LPS with acetic acid aspreviously described [8, 9]. The B-subunit of Shigella toxin I (StxB1)was synthesized by Vibrio cholerae strain 0395-N1 (pSBC32 containing theStxB1 gene) and purified by affinity chromatography[20, 21]. SDS 7% PAGEof StxB1 showed one major band at 9 kDa and a faint band with slightlyhigher molecular weight.

For conjugation, O157 O-SP was bound to StxB1 directly by treatment with1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) or bycarbodiimide mediated condensation with adipic acid hydrazide linker[29, 30]. For direct conjugation, CDAP (100 mg/ml in acetonitrile) wasadded to O-SP in saline (5 mg/ml) at 0.3/1 (wt/wt) at room temperature,pH 5.8 to 6. 60 μL of 0.2 M triethylamine (TEA) added to bring the pH to7.0 for 2 minutes. An equal weight of StxB1 was added to the CDAPtreated O-SP and the pH maintained at 8.0 to 8.5 for 2 hours. Thereaction mixture was passed through a 1.5×90 cm Sepharose 6B column in0.2M NaCl, the void volume fractions collected, and designated asOSP-StxB1.

Conjugate using a linker, adipic acid dihydrazide (ADH) was prepared asdescribed previously [8, 30]. Briefly after addition of TEA in the aboveprocedure, an equal volume of 0.8 M ADH in 0.5 M NaHCO₃ was added andthe pH maintained at 8.0 to 8.5 for 2 hours. The reaction mixture wasdialyzed against saline overnight at 4° C. and passed through a 2.5×31cm P10 column in water. The void volume fractions were pooled,freeze-dried, and designated as OSP-AH. OSP-AH (10 mg), dissolved in 2ml of saline, was added to an equal weight of StxB1 and the pH broughtto 5.1. The reaction mixture was put on ice and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added to 0.05Mand the pH maintained at 5.1 to 5.5 for 2 hours. The reaction mixturewas passed through a 1.5×90 cm Sepharose 6B column in 0.2 M NaCl, thevoid volume fractions collected and designated as OSP-AH-StxB1. Doubleimmunodiffusion and ELISA were performed as described [8].

Female general purpose mice (n=10/group) were injected subcutaneouslywith saline or one of the conjugates containing 2.5 μg saccharide ondays 0, 14, and 28. The mice were exsanguinated 7 days after eachinjection. Pooled sera from hyperimmunized mice were used as referenceand assigned 100 ELISA units for IgG and IgM respectively.Neutralization of Stx1 and Stx2 toward HeLa cells was measured usingHeLa (CCL-2) cell monolayers in 96-well flat-bottom microtiter plates[21]. Each well was seeded with 1-6×10⁴ cells in 0.1 ml. Monolayers wereestablished by overnight incubation in 5% CO₂. Toxin neutralization wasdetermined by incubating dilutions of mouse serum with Stx-I or Stx-IIat a final concentration of 100 μg/ml. The serum and toxin mixture wasincubated at room temperature for 30 minutes and 0.1 ml was added toeach well. Following incubation overnight, the surviving cells weredetermined spectro-photometrically using the crystal violet stainingmethod of Gentry and Dalrymple[31]. Toxin neutralization was determinedfrom a dose response curve of either Shiga toxin on each 96-well plate.Bactericidal activity was assayed as described [8, 10].

Results with O157 O-SP—STXB1 Conjugates

Derivatization of O-SP with adipic acid dihydrazide was 3.1% (wt/wt),similar to previous E. coli O157 preparations [8]. Thesaccharide/protein ratios (wt/wt) were about 0.5 for both conjugates.The yields, based on saccharide in the conjugates, were 2.3% forOSP-StxB1 and 3.4% for OSP-AH-StxB1. A single line of precipitation indouble immunodiffusion was formed by rabbit anti-Stx1 and mousehyperimmune anti-O157 reacted against either conjugate.

After three injections, both conjugates elicited statisticallysignificant rises of IgG and IgM anti-LPS (Table 6). The geometric mean(GM) anti-LPS level elicited by OSP-StxB1 was 0.63 for IgG and 0.14 forIgM and for O-SP-AH-StxB1 were 1.7 for IgG and 0.25 for IgM: thedifferences between two conjugates were not statistically significant.TABLE 6 Geometric mean IgG and IgM serum LPS antibody levels andneutralization titers against Shiga toxin 1 elicited in mice byconjugates of Escherichia coli O157 O-SP with StxB1. NeutralizationTiter (%)† Anti-LPS (ELISA)* Serum Dilution Immunogen IgG IgM 1:1001:1000 1:10,000 Saline <0.05 <0.05    0‡ 0 0 OSP-AH-StxB1 1.7 0.25 >9990 34 OSP-StxB1 0.63 0.14 >99 98 70*Geometric mean of sera from 10 mice. Expressed in ELISA units usingpooled hyperimmune mouse sera as reference (100 units for IgG and IgMrespectively).†Geometric mean (n = 10) neutralization titer determined with Stx1 andHeLa cells,‡No neutralization at 1/100 dilution.

Sera from mice injected with saline or human sera from volunteersinjected with E. coli O157 O-SP-rEPA conjugates showed no neutralizationto Stx1 or to Stx2. Sera from mice injected 3 times with either of theO157 O-SP-StxB1 conjugates showed complete neutralization of Stx1 at{fraction (1/100)} dilution. At {fraction (1/1,000)} dilution, the GM ofneutralization titer was 90% for OSP-AH-StxB1 and 98% for OSP-StxB1. At{fraction (1/10,000)} dilution, the sera from mice injected withOSP-StxB1 had a significantly higher neutralization titer (70%) than thesera elicited by O-SP-AH StxB1 (34%). None of the sera from miceinjected with either conjugate showed neutralization against Stx2. Bothconjugates elicited levels of bactericidal antibodies against E. coliO157 that were roughly proportional to the content of IgG anti-LPS; thisactivity was removed by absorption with E. coli O157 LPS.

DISCUSSION

The O-SP of E. coli O157 LPS is a linear copolymer composed of thetetrasaccharide repeat unit:(→3)-α-D-GalpNAc-(1→2)-α-D-PerpNAc-(1→3)-α-L-Fucp-(1→4)-β-D-Glcp-(1→).It is non-immunogenic, probably due to its comparatively low molecularweight. As with other polysaccharides, its immunogenicity is increasedby binding it to proteins to form a conjugate. Of the three conjugatesof the present invention shown in Table 1, none elicited fever orsignificant local reactions in human volunteers, and all volunteersresponded with a ≧4-fold rise in serum IgG anti-E. coli O157 LPS thatwas sustained 26 weeks after injection. (Re-injection of the E. coliO157 O-SP conjugates was not attempted because of the failure of otherpolysaccharide conjugates to induce a booster response in adults.)

These volunteers, like most adults, had low levels of “natural” serumanti E. coli O157 LPS probably induced by cross-reacting antigens [32,33, 34, 35]. This is typical for other bacterial pathogens as well.Higher levels of anti-O157 LPS antibodies are found in patients withHUS, and in individuals involved in raising cattle in certain areas,probably as a result of previous contact with these organisms. Althoughthe unusual monosaccharide perosamine is found in the O-SP of both E.coli O157 and V. cholerae O1, we have not been able to detect across-reaction between human antibodies to these two antigens. Theconjugate prepared from the O-SP obtained by acetic acid hydrolysis(O-SP-rEPA) elicited significantly higher levels of anti-O 157 LPS atfour weeks than did conjugates prepared with hydrazine-treated LPS. TheLPS and ETA antibody levels, however, at 26 weeks post-injection weresimilar in all three groups (Table 1). As reported for patients withshigellosis and for adults vaccinated with Shigella conjugates, serumIgG anti-LPS rose to the highest level and was the most sustained of thethree serum immunoglobulins [13, 15, 34, 36, 37]. Similar results wereobtained in mice for the IgG anti-LPS responses elicited by E. coli O111conjugates [38].

The protective action of existing vaccines may be due to the inductionof a critical level of specific IgG antibodies that, in many cases,inactivate the inoculum of the pathogen on epithelial surfaces includingthe intestine [39, 40]. It is not commonly appreciated that serum IgG isa major immunoglobulin component of secretory fluids including that ofthe small intestine. As has been observed in mice [8], all threeconjugates induced IgG anti-LPS with bactericidal activity in thevolunteers (Table 2). Serum IgG anti-polysaccharide is the major, if notthe sole host component, that confers immunity induced by theseconjugates. Accordingly, it should be possible to standardize thepotency of E. coli O157 conjugates by chemical assay and by measurementof serum IgG anti-polysaccharide as has been done for Haemophilusinfluenzae type b conjugate vaccines.

The 1995 outbreak of E. coli O157 infection in Japan lasted severalmonths, partly due to the failure to identify the bacterial sources[41]. Most of the volunteers (81%) responded with nearly a 10-foldincrease in IgG anti-LPS 1 week after vaccination, indicating that thevaccine of this invention could serve to control E. coli O157 infectionduring an outbreak. Another use for the E. coli O157 conjugates of thisinvention would be to prepare high-titered IgG anti-LPS globulin forprophylaxis and treatment of case contacts during an outbreak. It hasbeen suggested that antibiotic treatment of patients increases theincidence of HUS, possibly by causing lysis of the E. coli O157 withrelease of additional Shiga toxins. Clinical and experimental data pointto LPS as the pathogenic agent for HUS and the other extraintestinallesions following infection with enteric Gram-negative pathogens [42,43]. There is also a suggestion of a direct role of Shiga toxins onrenal tissue involvement in HUS [44]. The present invention provides asolution to this problem in the form of a conjugate of E. coli O157 O-SPwith the B subunit of Shiga toxin 1. In mice, this conjugate inducesboth serum IgG anti-LPS and neutralizing antibodies to Shiga toxin 1.

The data show that the various E. coli O157 LPS-protein conjugatesdisclosed herein will generate high antibody levels in humans (i.e.,approximately 5-10 times more IgG in humans than in mice) and highneutralization antibody titers in humans (i.e., 10³ to 10⁴ in humans asopposed to 102 in mice). The data also show that the various E. coliO157 LPS-protein conjugates disclosed herein will generate a greaterthan 4-fold rise in IgG antibody levels in about 80% of human subjectsone week after a single injection and in all human subjects 4 weeksafter a single injection.

REFERENCES AND NOTES

-   1. For reviews, see:    -   (a) J. B. Robbins, R. Schneerson, S. Szu, V. Pozsgay, In:        Vaccinia, vaccinations and vaccinology: Jenner, Pasteur and        their successors (Ed.: S. Plotkin, B. Fantini), Elsevier,        Paris, p. 135-143 (1996).    -   (b) R. K. Sood, A. Fattom, V. Pavliak, R. B. Naso, Drug        Discovery Today, 1, 381-387 (1996).    -   (c) H. J. Jennings, R. K. Sood, In Neoglycoconjugates.        Preparation and Applications (Eds. Y. C. Lee, R. T. Lee),        Academic Press, New York, pp. 325-371 (1994).-   2. K. Landsteiner, The specificity of serological reactions, Harvard    University Press, Cambridge, (1970).-   3. R. Schneerson, O. Barrera, A. Sutton, J. B. Robbins, J. Exp. Med.    1980, 152, 361-376.-   4. J. B. Robbins, R. Schneerson, P. Anderson, D. H. Smith, J. Am    Med. Assoc. 1996, 276, 1181-1185.-   5. For example:    -   (a) Cohen, D., et al., Lancet, 349, 155-0159 (1997).    -   (b) Cohen, D., et al., Infect. Immun., 64, 4074-4077 (1997).-   6. Riley. L. W., et al., N. Engl. J. Med., 308, 681-685 (1983).-   7. Takeda, Y., World Health Statistics Quarterly, 50, 74-80 (1997)-   8. Konadu et al., Infection & Immunity, 62, 5048-5054 (1994)-   9. Robbins, J. B., et al., Clin. Infect. Dis., 15, 346-361 (1992)-   10. Konadu et al., Journal of Infectious Diseases, 177 383-387    (1998)-   11. Butler, T., Islam, M. R., Azad, M. A. K., Jones, P. K., J.    Pediatr., 110, 894-897 (1987)-   12. Proulx, F., et al., J. Pediatr., 121, 299-303 (1992).-   13. Cohen, D, C. Block, M. S. Green, G. Lowell, and I. Ofek, J.    Clin. Microbiol., 27, 162-167 (1989).-   14. Cohen, D., M. S. Green, C. Block, T. Roauch, and I. Ofek, J.    Infect. Dis., 157, 1068-1071 (1988).-   15. Robbins, J. B., and R. Schneerson, J. Infect. Dis., 161, 821-832    (1990).-   16. Taylor, D. N., et al., Infect. Immun., 61, 3678-3687 (1993).-   17. Cohen, D., S. Ashkenazi, et al., Lancet, 349, 155-159 (1997).-   18. Cryz, S. J., et al., J. Infect. Dis., 163, 1040-1045 91991).-   19. Weinstein, D. L., Jackson, M. P., Perera, L. P., Holmes, R. K.,    O'Brien, A. D., Infect. Immun., 57, 3743-3750 (1989)-   20. Acheson, et al., Infect. Immun., 61, 1098-1104 (1993).-   21. Pozsgay, V., Trinh, L., Shiloach, J., Robbins, J. B.,    Donohue-Rolfe A, Calderwood S B., Bioconjugate. Chem., 7, 45-55    (1996).-   22. Fattom, A., C. Lue, S. C. Szu, J. Mestecky, G. Schiffinan, D. A.    Bryla, W. F. Vann, D. Watson, L. M. Kimzey, J. B. Robbins, and R.    Schneerson, Infect. Immun., 58, 2309-2312 (1990).-   23. Devi, S. J., J. B. Robbins and R. Schneerson., Proc. Natl. Acad.    Sci. USA 88: 7175-7179, 1991 (1992).-   24. Szu, S. C., X. Li, A. L. Stone, and J. B. Robbins, Infect.    Immun. 59 4555-4561 (1991).-   25. Szu, S. C., A. L. Stone, J. D. Robbins, R. Schneerson, and J. B.    Robbins, J. Exp. Med. 166 1510-1524 (1987).-   26. C. Chu, et al., Infect. Immun., 59, 4450-4458 (1991).-   27. Kohn, J., Wilchek, M., FEBS Letters, 154, 209 (1983).-   28. Aleksic, S., Karch, H., Bockemühl, J., Int. J. Med. Microbiol.,    276, 221-230 (1992).-   29. Lees, A., Nelson, B., Mond, J. J., Vaccine., 14, 190-198 (1995).-   30. Konadu, E., Shiloach, J., Bryla, D. A., Robbins, J. B., Szu, S.    C., Infect. Immun., 64, 2709-2715 (1996).-   31. Gentry M., Dalrymple J. M., J. Clin. Micro., 12, 361-366 (1980).-   32. Chart, H, Rowe, B., Lancet, 341, 1282 (1993).-   33. Robbins J. B., Schneerson R., J Infect Dis., 161, 821-832    (1990).-   34. Greatorex J. S., Thorni G. M., J Clin Microbiol., 32, 1172-1178    (1994).-   35. Cohen, D., et al., Infect Immun., 64, 4074-4077 (1996).-   36. Ekwall E, et al., Serodiag. Immunother. Infect. Dis., 2, 171-182    (1988).-   37. Cohen D., et al., Infect Immun., 64, 4074-4077 (1996).-   38. Gupta, R. K., Egan W, Bryla D A, Robbins J B, Szu S C., Infect.    Immun., 63, 2805-2810 (1995).-   39. Farmer, J. J., et al., J Clin Microbiol., 21, 46-76 (1985).-   40. Chart, H., Scotland, S. M., Rowe, B., J Clin Microbiol.,    27,285-290 (1989).-   41. Watanabe, H., Wada, A., Inagak, Y., Tarnura, K., Lancet, 348,    831-832 (1996).-   42. Koster, F, et al., N. Engl. J. Med., 298, 927-933 (1978).-   43. Jalkanen, K. S., et al., Lancet, 1,685-688 (1990).-   44. Pickering, L. K., Obrig, T. G., Stapleton, F. B., Pediatr.    Infect. Di.s J, 13, 459-476 (1994).

All of the references referred to above are hereby incorporated byreference in their entirety.

1. A pharmaceutical composition, comprising: (1) about 5 μg to about 50μg of E. coli O157 0-specific polysaccharide covalently bound to a Bsubunit of Shiga toxin 1, a B subunit of Shiga toxin 2, a non-toxicShiga toxin 1 holotoxin, or a non-toxic Shiga toxin 2 holotoxin; and (2)a pharmaceutically acceptable carrier.
 2. The pharmaceutical compositionof claim 1, further comprising a linker.
 3. The pharmaceuticalcomposition of claim 1, comprising the B subunit of Shiga toxin
 1. 4.The pharmaceutical composition of claim 1, comprising the B subunit ofShiga toxin
 2. 5. The pharmaceutical composition of claim 1, comprisingthe non-toxic Shiga toxin 1 holotoxin.
 6. The pharmaceutical compositionof claim 1, comprising the non-toxic Shiga toxin 2 holotoxin.
 7. Thepharmaceutical composition of claim 1, further comprising an adjuvant.8. A method for inducing an immune response in a mammal against E. coliO157, comprising administering to the mammal a therapeutically effectiveamount of the pharmaceutical composition according to claim 1, therebyinducing an immune response.
 9. The method of claim 8, wherein theimmune response comprises induction of serum antibodies that havebacteriostatic or bactericidal activity against E. coli O157 and thatneutralize Shiga toxin 1 or Shiga toxin
 2. 10. The method of claim 8,wherein the mammal is a human.
 11. The method of claim 10, wherein thehuman is under five years of age.
 12. A composition, comprising isolatedhuman antibodies immunoreactive with both E. coli O157 O-specificpolysaccharide and with a toxin, wherein the toxin comprises the Bsubunit of Shiga toxin 1 or the B subunit of Shiga toxin
 2. 13. Thecomposition of claim 12, wherein the composition is selected from humanplasma, human serum, or human immunoglobulin fraction.
 14. A method forinducing an immune response in a human against E. coli O157, comprisingadministering to the human a therapeutically effective amount of thecomposition according to claim 12, thereby inducing an immune response.15. The method of claim 14, wherein the human is under five years ofage.
 16. The method of claim 14, comprising administering about 1 mg/kgto about 10 mg/kg of the isolated human antibodies.